Project 3 Title: Gene Therapy for Lung Disease in a Ferret Model of Cystic Fibrosis Project Summary The development of gene therapies to the CF lung has met with many challenges over the past two decades. The greatest of these has been a lack of animal models that reproduce the human CF lung phenotype, which has been solved by the creation of CF models in ferret and pig. These models will provide insights into the basic cell biology of the disease and facilitate the development of effective treatments, including gene therapy. This proposed project will focus on evaluating two gene therapy approaches in the CF ferret model and on elucidating key aspects of airway cell biology that will direct choices of the gene therapy approaches used. CF ferrets have two lung infection phenotypes that will be useful in developing therapies for CF?rapid, lethal neonatal infections during the first 1-3 weeks of life; and slower, chronic bacterial infections thereafter, typically leading to death by 3-5 months of life. We will use these robust lung phenotypes to develop lentivirus- and recombinant adeno-associated virus (rAAV)-based gene therapies for CF lung disease. The preliminary data presented, and vectors chosen for study, in this project are the product of the first two years of an R01 award that will transition into this PPG. Important goals of this project include: 1) the development and analysis of miRNA detargeting approaches to regulate cellular expression patterns of a lentiviral CFTR transgene and limit the phenotypic consequences of ectopic CFTR expression in multi-potent airway progenitor cells, 2) the generation of rAAV-CFTR vectors that are capable of reversing lung disease in the CF ferret model and that are also compatible with both the packaging limitations of the rAAV genome and cellular expression patterns required for efficient complementation of CFTR function, and 3) identification of the cell types in the lung that must be targeted for effective complementation of CF lung disease. The third goal of the proposal draws on the unique ability of the Engelhardt laboratory to rapidly generate transgenic ferrets that express recombinant fCFTR in biologically relevant cell types within the lung. Thus, this proposal uses novel viral targeting methods to address cellular mechanisms relevant to improving gene therapies to the airway, while also tackling difficult cell biology questions about the pathogenesis and treatment of CF lung disease using gene therapy. This research is expected to significantly enhance the field's ability to develop effective therapies for CF lung disease.